Establishment and Optimization of an Efficient Peanut Genetic Transformation System

INTRODUCTION: To establish an efficient Agrobacterium-mediated genetic transformation system for peanuts and lay a foundation for the study of peanut gene functions and variety breeding. RATIONALE: In this study, 11 peanut varieties were selected, and the cotyledonary leaflets from the variety with the highest bud cluster induction rate were screened out as experimental materials. By screening and optimizing influencing factors such as Agrobacterium strains, the optical density (OD) value of the bacterial suspension, the concentration of acetosyringone (AS), the concentration of surfactants, the infection method and duration, and the co-culture time, transgenic plants of peanut cotyledonary leaflets were obtained. RESULTS: The results showed that using the embryo leaflet of Huayu9133 as the receptor, the recombinant Agrobacterium strains containing eGFP (green fluorescent protein) and GUS (β-glucosidase) were used to infect and transform, respectively. The results showed that when the infection solution was MS liquid+LBA4404 strain+100 μmol∙L–1 AS+150 mg∙L–1 surfactant Silwet-77, bacterial solution OD600 was 0.7, and the infection method was vacuuming for 15 min+soaking for 20 min+co-culture for 4 d, the peanut conversion rate was the highest. The positive rates of CaMV35S: eGFP and AhUBQ4: GUS were 52.67% and 57.67%, respectively. The transgenic plants were induced by tissue culture method. The transgenic plants containing eGFP protein were identified as transgenic positive plants by eGFP green fluorescence and PCR detection, and the transgenic plants containing GUS protein were identified as transgenic positive plants by GUS staining and PCR detection. CONCLUSION: This experiment successfully established and optimized the peanut genetic transformation system, which provided a solid foundation for the study of peanut gene function, the cultivation of resistant varieties, quality improvement and biotechnology research. Fluorescence schematic diagram of CaMV35S：eGFP infection material (a: Regeneration bud cluster under the bright field (bar=500 μm); b: Regeneration bud cluster fluorescence (bar=500 μm); c: Regeneration plantlet under the bright field (bar=2 mm); d: Regeneration plantlet fluorescence (bar=2 mm))